Methods and Apparatus for Analyzing Samples and Collecting Sample Fractions

ABSTRACT

Methods and apparatus for analyzing a sample using at least one detector are disclosed.

FIELD OF THE INVENTION

The present invention is directed to methods and apparatus for analyzingsamples and collecting sample fractions with a chromatography system.

BACKGROUND OF THE INVENTION

There is a need in the art for methods of efficiently and effectivelyanalyzing samples and collecting sample fractions with a chromatographysystem. There is also a need in the art for an apparatus capable ofeffectively analyzing samples and collecting sample fractions.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of methods for analyzingsamples and collecting sample fractions with a chromatography system.The disclosed methods provide a number of advantages over known methodsof analyzing samples. For example, the disclosed methods of the presentinvention may utilize a splitter pump or a shuttle valve to activelycontrol fluid flow through at least one detector so that processvariables (e.g., flow restrictions, total flow rate, temperature, and/orsolvent composition) do not negatively impact the fluid flow through theat least one detector. The disclosed methods of the present inventionmay also utilize two or more detectors to provide a more completeanalysis of a given sample, as well as collection of one or more samplefractions in response to one or more detector signals from the two ormore detectors.

The present invention is directed to methods of analyzing samples andcollecting sample fractions. In one exemplary embodiment, the method ofanalyzing a sample comprises the steps of generating a composite signalfrom two or more detectors in a liquid chromatography system, thecomposite signal comprising a detection response component from eachdetector; and collecting a new sample fraction in a fraction collectorin response to a change in the composite signal. In one embodiment, thecomposite signal may comprise (i) a detection response component from atleast one optical absorbance detector (e.g., an UV detector) and (ii) adetection response component from at least one evaporative particledetector. In one embodiment, chromophoric or non-chromophoric solventsmay be utilized in the chromatography system as the carrier fluid. Inanother embodiment, the composite signal may comprise (i) a detectionresponse component comprising two or more detector responses from anoptical absorbance detector (e.g., an UV detector) at two or morespecific optical wavelengths and (ii) a detection response componentfrom an evaporative particle detector.

In a further exemplary embodiment according to the present invention,the method of analyzing a sample using chromatography comprises thesteps of using at least one detector to observe the sample thatcomprises at least one non-chromaphoric analyte compound; and collectinga new sample fraction in a fraction collector in response to a change ina detector response of the non-chromaphoric compound. The sample mayinclude numerous different chromaphoric and non-chromaphoric compounds.In addition, the mobile phase that carries the sample may include one ormore chromaphoric or non-chromaphoric compounds.

In another embodiment, universal carrier fluid may be utilized in thechromatography system, including volatile liquids and various gases. Ina further embodiment, a non-destructive detector (e.g., RI, UV detector,etc.) may be combined with a destructive detector (e.g., evaporativeparticle detector, mass spectrometer, spectrophotometer, emissionspectroscopy, NMR, etc.), which enables detection of various compoundspecific properties of the sample, such as, for example, the chemicalentity associated with the peak.

In a further exemplary embodiment, the method of analyzing a samplecomprises the steps of using at least one detector to observe the sampleat two or more specific optical wavelengths; and collecting a new samplefraction in a fraction collector in response to (i) a change in adetector response at a first wavelength, (ii) a change in a detectorresponse at a second wavelength, or (iii) a change in a compositeresponse represented by the detector responses at the first and secondwavelengths. A change in a given detector response may include, but isnot limited to, a change in a detector response value, reaching orexceeding a threshold detector response value, a slope of the detectorresponse value over time, a threshold slope of the detector responsevalue over time, a change in a slope of the detector response value overtime, a threshold change in a slope of the detector response value overtime, or any combination thereof. In this embodiment, the method maycomprise using n sensors in at least one detector to observe n specificwavelengths across a range of an absorbance spectrum, wherein n is aninteger greater than 1; and collecting a new sample fraction in thefraction collector in response to (i) a change in any one of n detectorresponses from the n sensors, or (ii) a change in a composite responserepresented by the n detector responses.

In yet a further exemplary embodiment, the method of analyzing a samplecomprises the steps of providing a liquid chromatography systemcomprising (i) a chromatography column, (ii) a tee having a first inlet,a first outlet and a second outlet, (iii) a fraction collector in fluidcommunication with the first outlet of the tee, and (iv) a detector influid communication with the second outlet of the tee; and activelycontrolling fluid flow through the detector via (v) a splitter pumppositioned in fluid communication with the second outlet of the tee andthe detector. In other exemplary embodiments, a shuttle valve may beused in place of the tee and splitter pump to actively control fluidflow through to at least one detector. In an exemplary embodiment, theshuttle valve is a continuous flow shuttle valve with the ability toremove very small sample volumes from the sample stream.

In an even further exemplary embodiment of the present invention, amethod of analyzing a sample of fluid using chromatography includes thesteps of providing a first fluid of effluent from a chromatographycolumn; providing a second fluid to carry the sample of fluid to atleast one detector; using a shuttle valve to remove an aliquot sample offluid from the first fluid and transfer the aliquot to the second fluidwhile maintaining a continuous path of the second fluid through theshuttle valve; using at least one detector to observe the aliquot sampleof fluid; and collecting a new sample fraction of the first fluid in afraction collector in response to a change in a detector response. Inone embodiment, a continuous flow path of the first fluid through theshuttle valve is maintained when the aliquot sample of fluid is removedfrom the first fluid. In another embodiment, continuous flow paths ofboth the first fluid and the second fluid through the shuffle valve aremaintained when the aliquot sample of fluid is removed from the firstfluid and transferred to the second fluid.

In another exemplary embodiment according to the present invention, amethod of analyzing a sample of fluid using chromatography includes thesteps of providing a first fluid comprising the sample; using a shuttlevalve to remove an aliquot sample of fluid from the first fluid withoutsubstantially affecting flow properties of the first fluid through theshuttle valve; using at least one detector to observe the aliquot sampleof fluid; and collecting a new sample fraction of the first stream in afraction collector in response to a change in at least one detectorresponse. The flow of the first fluid through the shuttle valve may besubstantially laminar, due to the first fluid path or channel beingsubstantially linear or straight through at least a portion of thevalve. In a further exemplary embodiment, the pressure of the firstfluid through the shuttle valve remains substantially constant and/or itdoes not substantially increase. In another embodiment, the flow rate ofthe first fluid may be substantially constant through the shuttle valve.In an alternative embodiment, a second fluid is utilized to carry thealiquot sample of fluid from the shuttle valve to the detector(s). Theflow of the second fluid through the shuttle valve may be substantiallylaminar due to the second fluid path or channel being substantiallylinear or straight through at least a portion of the valve. In anexemplary embodiment, the pressure of the second fluid through theshuttle valve is substantially constant and/or it does not substantiallyincrease. In another embodiment, the flow rate of the second fluid maybe substantially constant through the shuttle valve.

In a further exemplary embodiment, the method of analyzing a samplecomprises the steps of providing a non-destructive system liquidchromatography system comprising (i) a chromatography column, (ii) twoor more non-destructive detectors (e.g., an optical absorbance detectorsuch as a UV detector) with no destructive detectors (e.g., a massspectrometer) present in the system, and (iii) a fraction collector influid communication with the two or more non-destructive detectors; andcollecting one or more sample fractions in response to detector signalsfrom the two or more non-destructive detectors.

In another exemplary embodiment according to the present invention, amethod of analyzing a sample using flash chromatography includes thesteps of using an evaporative particle detector to observe the samplethat is capable of detecting individual compounds; and collecting a newsample fraction in a fraction collector in response to a change in adetector response of the compound, wherein the evaporative particledetector is the only detector utilized to analyze the sample. Theevaporative particle detector is capable of detecting chemicalcomposition, chemical structure, molecular weight, or other chemical orphysical properties. The detector may include an ELSD, CNLSD or massspectrometer.

In yet a further exemplary embodiment, the method of analyzing a samplecomprises the steps of generating a detector signal from at least onedetector in a liquid chromatography system, the detector signal beinggenerated in response to (i) the slope of a detector response as afunction of time (i.e., the first derivative of a detector response),(ii) a change in the slope of the detector response as a function oftime (i.e., the second derivative of the detector response), (iii)optionally, reaching or exceeding a threshold detector response value,or (iv) any combination of (i) to (iii) desirably comprising at least(i) or at least (ii); and collecting one or more sample fractions inresponse to at least one detector signal from the at least one detector.

In yet another exemplary embodiment, the method of analyzing a samplecomprises the step of collecting a sample fraction in a fractioncollector of a liquid chromatography system, wherein the fractioncollector is operatively adapted to (i) recognize, receive and processone or more signals from at least one detector, and (ii) collect one ormore sample fractions based on the one or more signals.

The present invention is also directed to an apparatus capable ofanalyzing a sample. In one exemplary embodiment, the apparatus foranalyzing a sample comprises system hardware operatively adapted togenerate a composite signal from two or more detectors in a liquidchromatography system, the composite signal comprising a detectionresponse component from each detector; and a fraction collectoroperatively adapted to collect a new sample fraction in response to achange in the composite signal.

In another exemplary embodiment, the apparatus for analyzing a samplecomprises at least one detector operatively adapted to observe two ormore specific optical wavelengths (e.g., UV wavelengths); and a fractioncollector operatively adapted to collect a new sample in response to (i)a change in a detector response at a first wavelength, (ii) a change ina detector response at a second wavelength, or (iii) a change in acomposite response represented by the detector responses at the firstand second wavelengths. As discussed above, a change in a given detectorresponse may include, but is not limited to, a change in a detectorresponse value, reaching or exceeding a threshold detector responsevalue, a slope of the detector response value over time, a thresholdslope of the detector response value over time, a change in a slope ofthe detector response value over time, a threshold change in a slope ofthe detector response value over time, or any combination thereof.

The at least one detector may together comprise n sensors to observe aspecific wavelengths across a range of an absorbance spectrum, wherein nis an integer greater than 1, and the fraction collector is operativelyadapted to collect a new sample in response to (i) a change in any oneof a detector responses from the n sensors, or (ii) a change in acomposite response represented by the n detector responses. In oneembodiment, the apparatus comprises a single UV detector comprising nsensors alone or in combination with one or more additional detectors.

In yet a further exemplary embodiment, the apparatus for analyzing asample comprises system hardware that enables generation of a detectorsignal from at least one detector in a liquid chromatography system, thedetector signal being generated in response to (i) the slope of adetector response as a function of time (i.e., the first derivative of adetector response), (ii) a change in the slope of the detector responseas a function of time (i.e., the second derivative of the detectorresponse), (iii) optionally, reaching or exceeding a threshold detectorresponse value, or (iv) any combination of (i) to (iii) desirablycomprising at least (i) or at least (ii). The apparatus may furthercomprise a fraction collector operatively adapted to collect one or moresample fractions in response to the detector signal from the at leastone detector.

In another exemplary embodiment according to the present invention, anapparatus for analyzing a sample using chromatography includes at leastone detector that are capable of detecting chromaphoric andnon-chromaphoric analyte compounds in the sample; and a fractioncollector that is capable of responding to a change in a detectorresponse of the non-chromaphoric compound. The sample may includenumerous different chromaphoric and non-chromaphoric compounds. Inaddition, the mobile phase that carries the sample may include one ormore chromaphoric or non-chromaphoric compounds.

In yet a further exemplary embodiment, the apparatus for analyzing asample comprises (i) a chromatography column; (ii) a tee having a firstinlet, a first outlet and a second outlet; (iii) a fraction collector influid communication with the first outlet of the tee; (iv) a firstdetector in fluid communication with the second outlet of the tee; and(v) a splitter pump positioned in fluid communication with the secondoutlet of the tee and the first detector, the splitter pump beingoperatively adapted to actively control fluid flow through the firstdetector. In other exemplary embodiments, a shuttle valve may be used inplace of the tee and splitter pump to actively control fluid flowthrough to at least one detector. In an exemplary embodiment, theshuttle valve is a continuous flow shuttle valve.

In an even further embodiment according to the present invention, anapparatus for analyzing a sample of fluid using chromatography includesa first fluid path of effluent from a chromatography column orcartridge; at least one detector that is capable of analyzing the sampleof fluid; and a shuttle valve that transfers an aliquot sample of fluidfrom the first fluid path to the detector(s) without substantiallyaffecting the flow properties of fluid through the first fluid path. Theflow of the fluid through the first fluid path may be substantiallylaminar, due to the first fluid path or channel being substantiallylinear or straight through at least a portion of the valve. In a furtherexemplary embodiment, the pressure of the fluid through the first fluidpath remains substantially constant and/or it does not substantiallyincrease. In another embodiment, the flow rate of the fluid may besubstantially constant through the first fluid path. In an alternativeembodiment, a second fluid path is utilized to carry the aliquot sampleof fluid from the shuttle valve to the detector(s). The flow of fluidthrough the second fluid path may be substantially laminar due to thesecond fluid path or channel being substantially linear or straightthrough at least a portion of the valve. In an exemplary embodiment, thepressure of fluid through the second fluid path is substantiallyconstant and/or it does not substantially increase. In furtherembodiment, the flow rate of fluid may be substantially constant throughthe second fluid path.

In an even further exemplary embodiment, an apparatus for analyzing asample of fluid using chromatography includes a first fluid path ofeffluent from a chromatography column; a second fluid path that carriesthe sample of fluid to at least one detector that is capable ofanalyzing the sample; and a shuttle valve that transfers an aliquotsample of fluid from the first fluid path to the second fluid path whilemaintaining a continuous second fluid path through the shuttle valve. Inone embodiment, a continuous first flow path through the shuttle valveis maintained when the aliquot sample of fluid is removed from the firstfluid path. In another embodiment, continuous first and second flowpaths through the shuttle valve are maintained when the aliquot sampleof fluid is removed from the first fluid path and transferred to thesecond fluid path.

In a further exemplary embodiment, the apparatus for analyzing a samplecomprises (i) a chromatography column; (ii) two or more non-destructivedetectors with no destructive detectors within the system; (iii) afraction collector in fluid communication with the two or morenon-destructive detectors, the fraction collector being operativelyadapted to collect one or more sample fractions in response to one ormore detector signals from the two or more non-destructive detectors.

In an even further embodiment according to the present invention, anapparatus for analyzing a sample using flash chromatography includes anevaporative particle detector that is capable of detecting individualcompounds in the sample; and a fraction collector that is capable ofresponding to a change in a detector response of the detected compound,wherein the evaporative particle detector is the only detector utilizedto analyze the sample. The evaporative particle detector is capable ofdetecting chemical composition, chemical structure, molecular weight, orother physical or chemical properties. The detector may include an ELSD,CNLSD or mass spectrometer.

In yet another exemplary embodiment, the apparatus for analyzing asample comprises a fraction collector in a liquid chromatography system,the fraction collector being operatively adapted to (i) recognize,receive and process one or more signals from at least one detector, and(ii) collect one or more sample fractions based on the one or moresignals.

The methods and apparatus of the present invention may comprise at leastone detector. Suitable detectors include, but are not limited to,non-destructive detectors (i.e., detectors that do not consume ordestroy the sample during detection) such as UV, RI, conductivity,fluorescence, light scattering, viscometry, polorimetry, and the like;and/or destructive detectors (i.e., detectors that consume or destroythe sample during detection) such as evaporative particle detectors(EPD), e.g., evaporative light scattering detectors (ELSD), condensationnucleation light scattering detectors (CNLSD), ect., corona discharge,mass spectrometry, atomic adsorption, and the like. For example, theapparatus of the present invention may include at least one UV detector,at least one evaporative light scattering detector (ELSD), at least onemass spectrometer (MS), at least one condensation nucleation lightscattering detector (CNLSD), at least one corona discharge detector, atleast one refractive index detector (RID), at least one fluorescencedetector (FD), chiral detector (CD) or any combination thereof. In oneexemplary embodiment, the detector may comprise one or more evaporativeparticle detector(s) (EPD), which allows the use of chromaphoric andnon-chromaphoric solvents as the mobile phase. In a further embodiment,a non-destructive detector may be combined with a destructive detector,which enables detection of various compound specific properties,molecular weight, chemical structure, elemental composition andchirality of the sample, such as, for example, the chemical entityassociated with the peak.

The present invention is even further directed to computer readablemedium having stored thereon computer-executable instructions forperforming one or more of the method steps in any of the exemplarymethods described herein. The computer readable medium may be used toload application code onto an apparatus or an apparatus component, suchas any of the apparatus components described herein, in order to (i)provide interface with an operator and/or (ii) provide logic forperforming one or more of the method steps described herein.

These and other features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an exemplary liquid chromatography system of the presentinvention comprising a splitter pump to actively control fluid flow to adetector;

FIG. 2 depicts another exemplary liquid chromatography system of thepresent invention comprising a splitter pump and a detector;

FIG. 3A depicts an exemplary liquid chromatography system of the presentinvention comprising a shuttle valve and a detector;

FIGS. 3B-3C depict the operation of an exemplary shuttle valve suitablefor use in the present invention;

FIG. 4 depicts an exemplary liquid chromatography system of the presentinvention comprising a splitter pump and two detectors;

FIG. 5 depicts an exemplary liquid chromatography system of the presentinvention comprising two splitter pumps and two detectors;

FIG. 6 depicts an exemplary liquid chromatography system of the presentinvention comprising a shuttle valve and two detectors;

FIG. 7 depicts an exemplary liquid chromatography system of the presentinvention comprising two shuttle valves and two detectors;

FIG. 8 depicts an exemplary liquid chromatography system of the presentinvention comprising a splitter pump, an evaporative light scatteringdetector (ELSD), and an ultraviolet (UV) detector;

FIG. 9 depicts another exemplary liquid chromatography system of thepresent invention comprising a splitter pump, an ELSD and an UVdetector;

FIGS. 10A-10C depict the operation of an exemplary shuttle valvesuitable for use in the present invention; and

FIG. 11 depicts a chromatogram produced from the separation of a twocomponent mixture using an exemplary chromatography system of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

To promote an understanding of the principles of the present invention,descriptions of specific embodiments of the invention follow andspecific language is used to describe the specific embodiments. It willnevertheless be understood that no limitation of the scope of theinvention is intended by the use of specific language. Alterations,further modifications, and such further applications of the principlesof the present invention discussed are contemplated as would normallyoccur to one ordinarily skilled in the art to which the inventionpertains.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asolvent” includes a plurality of such solvents and reference to“solvent” includes reference to one or more solvents and equivalentsthereof known to those skilled in the art, and so forth.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperatures, processtimes, recoveries or yields, flow rates, and like values, and rangesthereof, employed in describing the embodiments of the disclosure,refers to variation in the numerical quantity that may occur, forexample, through typical measuring and handling procedures; throughinadvertent error in these procedures; through differences in theingredients used to carry out the methods; and like proximateconsiderations. The term “about” also encompasses amounts that differdue to aging of a formulation with a particular initial concentration ormixture, and amounts that differ due to mixing or processing aformulation with a particular initial concentration or mixture. Whethermodified by the term “about” the claims appended hereto includeequivalents to these quantities.

As used herein, the term “chromatography” means a physical method ofseparation in which the components to be separated are distributedbetween two phases, one of which is stationary (stationary phase) whilethe other (the mobile phase) moves in a definite direction.

As used herein, the term “liquid chromatography” means the separation ofmixtures by passing a fluid mixture dissolved in a “mobile phase”through a column comprising a stationary phase, which separates theanalyte (i.e., the target substance) from other molecules in the mixtureand allows it to be isolated.

As used herein, the term “mobile phase” means a fluid liquid, a gas, ora supercritical fluid that comprises the sample being separated and/oranalyzed and the solvent that moves the sample comprising the analytethrough the column. The mobile phase moves through the chromatographycolumn or cartridge (i.e., the container housing the stationary phase)where the analyte in the sample interacts with the stationary phase andis separated from the sample.

As used herein, the term “stationary phase” means material fixed in thecolumn or cartridge that selectively adsorbs the analyte from the samplein the mobile phase separation of mixtures by passing a fluid mixturedissolved in a “mobile phase” through a column comprising a stationaryphase, which separates the analyte to be measured from other moleculesin the mixture and allows it to be isolated.

As used herein, the term “flash chromatography” means the separation ofmixtures by passing a fluid mixture dissolved in a “mobile phase” underpressure through a column comprising a stationary phase, which separatesthe analyte (i.e., the target substance) from other molecules in themixture and allows it to be isolated.

As used herein, the term “shuttle valve” means a control valve thatregulates the supply of fluid from one or more source(s) to anotherlocation. The shuttle valve may utilize rotary or linear motion to movea sample from on fluid to another.

As used herein, the term “fluid” means a gas, liquid, and supercriticalfluid.

As used herein, the term “laminar flow” means smooth, orderly movementof a fluid, in which there is no turbulence, and any given subcurrentmoves more or less in parallel with any other nearby subcurrent.

As used herein, the term “substantially” means within a reasonableamount, but includes amounts which vary from about 0% to about 50% ofthe absolute value, from about 0% to about 40%, from about 0% to about30%, from about 0% to about 20% or from about 0% to about 10%.

The present invention is directed to methods of analyzing samples andcollecting sample fractions. The present invention is further directedto apparatus capable of analyzing samples and collecting samplefractions. The present invention is even further directed to computersoftware suitable for use in an apparatus or apparatus component that iscapable of analyzing samples and collecting sample fractions, whereinthe computer software enables the apparatus to perform one or moremethod steps as described herein.

A description of exemplary methods of analyzing samples and apparatuscapable of analyzing samples is provided below,

I. Methods of Analyzing Samples

The present invention is directed to methods of analyzing samples andcollecting sample fractions. The methods of analyzing a sample maycontain a number of process steps, some of which are described below.

A. Active Control of Fluid Flow to a Detector

In some embodiments of the present invention, the method of analyzing asample comprises a step comprising actively controlling fluid flow to adetector via a splitter pump or a shuttle valve. One exemplary liquidchromatography system depicting such a method step is shown in FIG. 1.As shown in FIG. 1, exemplary liquid chromatography system 10 comprises(i) a chromatography column 11, (ii) a tee 12 having a first inlet 21, afirst outlet 22 and a second outlet 23, (iii) a fraction collector 14 influid communication with first outlet 22 of tee 12, (iv) a firstdetector 13 in fluid communication with second outlet 23 of tee 12, and(v) a splitter pump 15 positioned in fluid communication with secondoutlet 23 of tee 12 and first detector 13.

In this exemplary system, splitter pump 15 actively controls fluid flowto first detector 13. As used herein, the phrase “actively controls”refers to the ability of a given splitter pump or shuttle valve tocontrol fluid flow through a given detector even though there may bechanges in fluid flow rate in other portions of the liquidchromatography system. Unlike “passive” flow splitters that merely splitfluid flow, the splitter pumps and shuttle valves used in the presentinvention control fluid flow to at least one detector regardless ofpossible fluctuations in fluid flow within the liquid chromatographysystem such as, for example, flow restrictions, total flow rate,temperature, and/or solvent composition.

The step of actively controlling fluid flow to a given detector maycomprise, for example, sending an activation signal to the splitter pumpor shuttle valve to (i) activate the splitter pump or shuttle valve,(ii) deactivate the splitter pump or shuttle valve, (iii) change one ormore flow and/or pressure settings of the splitter pump or shuttlevalve, or (iv) any combination of (i) to (iii), Suitable flow andpressure settings include, but are not limited to, (i) a valve position,(ii) splitter pump or shuttle valve pressure, (iii) air pressure to avalve, or (iv) any combinations of (i) to (iii). Typically, theactivation signal is in the form of, for example, an electrical signal,a pneumatic signal, a digital signal, or a wireless signal.

As shown in FIG. 1, in exemplary liquid chromatography system 10, thestep of actively controlling fluid flow to detector 13 comprises usingsplitter pump 15 to pump fluid from tee 12 into detector 13. In otherembodiments, the step of actively controlling fluid flow to a detectormay comprise using a splitter pump to pull fluid through a detector.Such a system configuration is shown in FIG. 2.

FIG. 2 depicts exemplary liquid chromatography system 20 compriseschromatography column 11; tee 12 having first inlet 21, first outlet 22and second outlet 23; fraction collector 14 in fluid communication withfirst outlet 22 of tee 12; first detector 13 in fluid communication withsecond outlet 23 of tee 12; and splitter pump 15 positioned so as topull fluid through detector 13 from second outlet 23 of tee 12.

In some desired embodiments, a shuttle valve, such as exemplary shuttlevalve 151 shown in FIGS. 3A-3C is used to actively control fluid flow toa detector such as detector 131. As shown in FIG. 3A, exemplary liquidchromatography system 30 comprises chromatography column 11; shuttlevalve 151 having chromatography cartridge inlet 111, fraction collectoroutlet 114, gas or liquid inlet 115 and detector outlet 113; fractioncollector 14 in fluid communication with fraction collector outlet 114of shuttle valve 151; first detector 131 in fluid communication withdetector outlet 113 of shuttle valve 151; and fluid supply 152 providingfluid to gas or liquid inlet 115 of shuttle valve 151.

In an even further exemplary embodiment of the present invention, amethod of analyzing a sample of fluid using chromatography includes thesteps of providing a first fluid of effluent from a chromatographycolumn; providing a second fluid to carry the sample of fluid to atleast one detector; using a shuffle valve to remove an aliquot sample offluid from the first fluid and transfer the aliquot to the second fluidwhile maintaining a continuous path of the second fluid through theshuttle valve; using at least one detector to observe the aliquot sampleof fluid; and collecting a new sample fraction of the first fluid in afraction collector in response to a change in a detector response. Inone embodiment, a continuous flow path of the first fluid through theshuttle valve is maintained when the aliquot sample of fluid is removedfrom the first fluid. In another embodiment, continuous flow paths ofboth the first fluid and the second fluid through the shuttle valve aremaintained when the aliquot sample of fluid is removed from the firstfluid and transferred to the second fluid.

In another exemplary embodiment according to the present invention, amethod of analyzing a sample of fluid using chromatography includes thesteps of providing a first fluid comprising the sample; using a shuttlevalve to remove an aliquot sample of fluid from the first fluid withoutsubstantially affecting flow properties of the first fluid through theshuttle valve; using at least one detector to observe the aliquot sampleof fluid; and collecting a new sample fraction of the first stream in afraction collector in response to a change in at least one detectorresponse. The flow of the first fluid through the shuttle valve may besubstantially laminar, due to the first fluid path or channel beingsubstantially linear or straight through at least a portion of thevalve. In a further exemplary embodiment, the pressure of the firstfluid through the shuttle valve remains substantially constant and/or itdoes not substantially increase. In another embodiment, the flow rate ofthe first fluid may be substantially constant through the shuttle valve.In an alternative embodiment, a second fluid is utilized to carry thealiquot sample of fluid from the shuffle valve to the detector(s). Theflow of the second fluid through the shuttle valve may be substantiallylaminar due to the second fluid path or channel being substantiallylinear or straight through at least a portion of the valve. In anexemplary embodiment, the pressure of the second fluid through theshuffle valve is substantially constant and/or it does not substantiallyincrease. In another embodiment, the flow rate of the second fluid maybe substantially constant through the shuttle valve.

FIGS. 3B-3C depict how a shuttle valve in one exemplary embodimentoperates within a given liquid chromatography system. As shown in FIG.3B, shuttle valve 151 comprises chromatography cartridge inlet 111,which provides fluid flow from a chromatography column (e.g., column 11)to shuttle valve 151; an incoming sample aliquot volume 116; fractioncollector outlet 114, which provides fluid flow from shuttle valve 151to a fraction collection (e.g., fraction collection 14); gas or liquidinlet 115, which provides gas (e.g., air, nitrogen, etc.) or liquid(e.g., an alcohol) flow through a portion of shuttle valve 151; outgoingsample aliquot volume 117; and detector outlet 113, which provides fluidflow from shuttle valve 151 to a detector (e.g., detector 131, such as aELSD).

As fluid flows through shuttle valve 151 from chromatography cartridgeto inlet 111 to fraction collector outlet 114, incoming sample aliquotvolume 116 is filled with a specific volume of fluid referred to hereinas sample aliquot 118 (shown as the shaded area in FIG. 3B). At adesired time, shuttle valve 151 transfers sample aliquot 118 withinincoming sample aliquot volume 116 into outgoing sample aliquot volume117 as shown in FIG. 3C. Once sample aliquot 118 is transferred intooutgoing sample aliquot volume 117, gas or liquid flowing from inlet 115through outgoing sample aliquot volume 117 transports sample aliquot 118to detector 131 (e.g., an ELSD) via detector outlet 113.

Shuttle valve 151 may be programmed to remove a sample aliquot (e.g.,sample aliquot 118) from a sample for transport to at least one detectorat a desired sampling frequency. In one exemplary embodiment, thesampling frequency is at least 1 sample aliquot every 10 seconds (or atleast 1 sample aliquot every 5 seconds, or at least 1 sample aliquotevery 3 seconds, or at least 1 sample aliquot every 2 seconds, or 1sample aliquot every 0.5 seconds, or at least 1 sample aliquot every 0.1seconds).

FIGS. 10A-C depict an exemplary shuttle valve of the present inventionand how it operates within a given liquid chromatography system. Asshown in FIG. 10A, shuttle valve 151 comprises chromatography cartridgeinlet 111, which provides fluid flow from a chromatography column (e.g.,column 11) to shuttle valve 151; channel 117 connecting inlet 111 tooutlet 114; an incoming sample aliquot volume 118 in dimple 116 ofdynamic body 119; fraction collector outlet 114, which provides fluidflow from shuttle valve 151 to a fraction collection (e.g., fractioncollection 14); gas or liquid inlet 115, which provides gas (e.g., air,nitrogen, etc.) or liquid (e.g., an alcohol) flow through shuttle valve151; outgoing sample aliquot volume 118 in dimple 116; channel 120connecting inlet 115 to outlet 113; and detector outlet 113, whichprovides fluid flow from shuttle valve 151 to a detector (e.g., detector131, such as a ELSD).

As fluid flows through shuttle valve 151 from chromatography cartridgeto inlet 111 to fraction collector outlet 114 via channel 117, incomingsample aliquot volume 118 in dimple 116 is filled with a specific volumeof fluid referred to herein as sample aliquot 118 (shown as the shadedarea in FIG. 10A). At a desired time, shuttle valve 151 transfers samplealiquot 118 within dimple 116 taken from channel 117 to channel 120 byrotating the dimple 116 in dynamic body 119 via dimple rotation path121. Once sample aliquot 118 is transferred into channel 120, gas orliquid flowing from inlet 115 through channel 120 transports samplealiquot 118 to detector 131 (e.g., an ELSD) via detector outlet 113.Another advantage of the shuttle valve of the present invention relatesto the fluidics design of the channels through the valve. In order tominimize backpressure in the chromatography system, the flow throughchannels 117 and 120 is continuous. This is accomplished by locatingchannels 117 and 120 in static body 122 such that no matter whatposition the dynamic body 119 is in, the flow through shuttle valve 151is continuous (as shown in FIG. 10B). As shown in FIG. 10A, at least aportion of the sample stream channel 117 and detector stream channel 120may be substantially planar or circumferential, which reduces turbulenceand further minimizes pressure increase through the valve. In addition,at least a portion of the sample stream channel 117 and detector streamchannel 120 may be substantially parallel to dimple 116 when contiguouswith it, which further limits turbulent flow and any increase inpressure in the valve. This includes those configurations that do notincrease pressure within the valve of more than 50 psi, preferably notmore than 30 psi, more preferably not more than 20 psi, and even morepreferably not more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 psi. Dimple116 is located in the dynamic body 119 and is in fluid communicationwith the face of the dynamic body that is contiguous with the staticbody 122, whereby when the dynamic body 119 is in a first position, thedimple 116 will be in fluid communication with the sample stream channel117, and when moved to a second position, the dimple 116 will be influid communication with the detector stream channel 120. The dimple 116may be of any shape but is depicted as a concave semi sphere, and it maybe or any size. In an exemplary embodiment, the dimple may be extremelysmall in size (e.g., less than 2000 nL, preferably less than about 500nL, more preferably less than about 100 nL, and even more preferablyless than about 1 nL, but may include any size from 1 nL to 2000 nL,which allows for rapid sampling. In addition, small dimple 116 sizeallows for a very short dimple rotation path 121, which significantlyreduces wear on the surfaces of the dynamic body 119 and the static body122 and results in a shuttle valve 151 having extended service lifebefore maintenance is required (e.g., more than 10 million cycles arepossible before service). Even though a rotary motion shuttle valve isdepicted in FIG. 10A-C, linear motion shuttle valves, or theirequivalent, may be employed in the present invention.

Shuttle valve 151 may be programmed to remove a sample aliquot (e.g.,sample aliquot 118) from a sample for transport to at least one detectorat a desired sampling frequency. In one exemplary embodiment, thesampling frequency is at least 1 sample aliquot every 10 seconds (or atleast 1 sample aliquot every 5 seconds, or at least 1 sample aliquotevery 3 seconds, or at least 1 sample aliquot every 2 seconds, or 1sample aliquot every 0.5 seconds, or at least 1 sample aliquot every 0.1seconds). This shuttle valve is further described in copending U.S.provisional patent application No. 61/200,814, the entire subject matterof which is incorporated herein by reference.

In another embodiment, universal carrier fluid, including volatileliquids and various gases, may be utilized in the chromatography systemto carry a sample to a detector. As shown in FIG. 3A, the carrier fluidfrom fluid supply 152 enters the shuttle valve 151 at inlet 115 where itpicks up sample aliquot 118 (shown in FIG. 10A) and then proceeds viaoutlet 113 to detector 131. The sample aliquot should not precipitate inthe carrier fluid of the valve or the associated plumbing may becomeblocked, or the sample will coat the walls of the flow path and some orall of the sample will not reach the detector. Sample composition inflash chromatography is very diverse, covering a large spectrum ofchemical compounds including inorganic molecules, organic molecules,polymers, peptides, proteins, and oligonucleotides. Solubility invarious solvents differs both within and between classes of compounds.Detector compatibility also constrains the types of carrier fluids thatmay be used. For example, for UV detection, the solvent should benon-chromaphoric at the detection wavelength. For evaporative particledetection (EPD) techniques (ELSD, CNLSD, Mass spec, etc.), the solventshould be easily evaporated at a temperature well below the sample'smelting point. In addition, the carrier fluid should be miscible withthe sample flowing between the valve inlet 111 and the fractioncollector outlet 114. For example, if hexane is used in one flow path,water may not be used in the other flow path because the two are notmiscible. All the above suggests the carrier fluid should be customizedeach time the separation solvents change. This is time consuming andimpractical. According to an exemplary embodiment of the presentinvention, using solvents that are miscible with organic solvents andwater, volatile, and non-chromaphoric, averts this problem. For example,a volatile, non-chromaphoric medium polarity solvent, such as isopropylalcohol (IPA), may be used as the carrier fluid. IPA is miscible withalmost all solvents, is non-chromaphoric at common UV detectionwavelengths, and is easily evaporated at low temperatures. In addition,IPA dissolves a broad range of chemicals and chemical classes. IPA isthus a suitable carrier fluid for virtually all sample types. Othercarrier fluids may include acetone, methanol, ethanol, propanol,butanol, isobutanol, tetrahydrofuran, and the like. In an alternativeexemplary embodiment, a gas may be utilized as the carrier fluid. Sampleprecipitation is not encountered because the sample remains in theseparation solvent, or mobile phase, through the shuttle valve andsubsequently through the detector. Likewise, the separation solvent, ormobile phase, never mixes with another solvent so miscibility is not anissue. Because the carrier is a gas, volatility is no longer an issue.In addition, most gasses are non-chromaphoric and compatible with UVdetection. When using gas as the carrier, the sample aliquot 118 isissued from the valve 151 to the detector 131 as discrete slugssandwiched between gas pockets 123 as shown in FIG. 10C. Using gas asthe carrier fluid has other advantages. For example, when used with anevaporative light scattering detector or other detection technique wherethe sample is nebulized, the gas may be used to transport the sample andnebulize the sample, eliminating the need for a separate nebulizer gassupply. In addition, because gas does not require evaporation, ambientdrift tube temperatures may be used eliminating the need for drift tubeheaters. A broader range of samples may be detected because those thatwould evaporate at higher temperatures will now stay in the solid orliquid state as they pass through the drift tube. A variety of gassesmay be used as the carrier gas including air, nitrogen, helium, hydrogenand carbon dioxide. Supercritical fluids may also be used, such assupercritical carbon dioxide.

B. Detection of a Sample Component within a Fluid Stream

The methods of the present invention may further comprise using at leastone detector to detect one or more sample components within a fluidstream. Suitable detectors for use in the liquid chromatography systemsof the present invention include, but are not limited to,non-destructive and/or destructive detectors. Suitable detectorsinclude, but are not limited to, non-destructive detectors (i.e.,detectors that do not consume or destroy the sample during detection)such as UV, RI, conductivity, fluorescence, light scattering,viscometry, polarimetry, and the like; and/or destructive detectors(i.e., detectors that consume or destroy the sample during detection)such as evaporative particle detectors (EPD), e.g., evaporative lightscattering detectors (ELSD), condensation nucleation light scatteringdetectors (CNLSD), etc., corona discharge, mass spectrometry, atomicadsorption, and the like. For example, the apparatus of the presentinvention may include at least one UV detector, at least one evaporativelight scattering detector (ELSD), at least one mass spectrometer (MS),at least one condensation nucleation light scattering detector (CNLSD),at least one corona discharge detector, at least one refractive indexdetector (RID), at least one fluorescence detector (FD), at least onechiral detector (CD), or any combination thereof. In one exemplaryembodiment, the detector may comprise one or more evaporative particledetector(s) (EPD), which allows the use of chromaphoric andnon-chromaphoric solvents as the mobile phase. In a further embodiment,a non-destructive detector may be combined with a destructive detector,which enables detection of various compound specific properties of thesample, such as, for example, the chemical entity, chemical structure,molecular weight, etc., associated with each chromatographic peak. Whencombined with mass spectrometer detection, the fraction's chemicalstructure and/or molecular weight may be determined at the time ofdetection, streamlining identification of the desired fraction. Incurrent systems the fraction's chemical identity and structure must bedetermined by cumbersome past-separation techniques.

Regardless of the type of detector used, a given detector provides oneor more detector responses that may be used to generate and send asignal to one or more components (e.g., a fraction collector, anotherdetector, a splitter pump, a shuttle valve, or a tee) within a liquidchromatography system as described herein. Typically, a change in agiven detector response triggers the generation and sending of a signal.In the present invention, a change in a given detector response thatmight trigger the generation and sending of a signal to one or morecomponents includes, but is not limited to, a change in a detectorresponse value, reaching or exceeding a threshold detector responsevalue, a slope of the detector response value over time, a thresholdslope of the detector response value over time, a change in a slope ofthe detector response value over time, a threshold change in a slope ofthe detector response value over time, or any combination thereof.

In some exemplary embodiments, the liquid chromatography system of thepresent invention comprises at least two detectors as shown in FIG. 4.Exemplary liquid chromatography system 40 shown in FIG. 4 compriseschromatography column 11; tee 12 having first inlet 21, first outlet 22and second outlet 23; fraction collector 14 in fluid communication withfirst outlet 22 of tee 12; first detector 13 in fluid communication withsecond outlet 23 of tee 12; splitter pump 15 actively controlling fluidflow to first detector 13 from second outlet 23 of tee 12; and seconddetector 16 in fluid communication with second outlet 23 of tee 12.

When two or more detectors are present, the liquid chromatography systemprovides more analysis options to an operator. For example, in exemplaryliquid chromatography system 40 shown in FIG. 4, a method of analyzing asample may comprise a step of sending one or more signals from firstdetector 13 (e.g., an ELSD) and/or second detector 16 (e.g., an opticalabsorbance detector such as an UV detector) to fraction collector 14instructing fraction collector 14 to collect a new sample fraction. Theone or more signals from first detector 13 and/or second detector 16 maycomprise a single signal from first detector 13 or second detector 16,two or more signals from first detector 13 and second detector 16, or acomposite signal from first detector 13 and second detector 16. Inexemplary liquid chromatography system 40 shown in FIG. 4, the method ofanalyzing a sample may further comprise a step of sending a signal fromsecond detector 16 to splitter pump 15 instructing splitter pump 15 toinitiate or stop fluid flow to first detector 13 in response to seconddetector 16 detecting a sample component in a fluid stream.

In other exemplary embodiments, the liquid chromatography system of thepresent invention comprises at least two detectors and at least twosplitter pumps as shown in FIG. 5. Exemplary liquid chromatographysystem 50 shown in FIG. 5 comprises chromatography column 11; first tee12 having first inlet 21, first outlet 22 and second outlet 23; firstdetector 13 in fluid communication with second outlet 23 of first tee12; first splitter pump 15 actively controlling fluid flow to firstdetector 13 from second outlet 23 of first tee 12; second tee 18 havingfirst inlet 31, first outlet 32 and second outlet 33; second detector 16in fluid communication with second outlet 33 of second tee 18; secondsplitter pump 17 actively controlling fluid flow to second detector 16from second outlet 33 of second tee 18; and fraction collector 14 influid communication with second outlet 32 of second tee 18.

As discussed above, the liquid chromatography systems of the presentinvention may comprise one or more shuttle valves in place or one ormore tee/splitter pump combinations to actively control fluid flow to atleast one detector as exemplified in FIGS. 6-7. As shown in FIG. 6,exemplary liquid chromatography system 60 comprises chromatographycolumn 11; shuttle valve 151 having chromatography cartridge inlet 111,fraction collector outlet 114, gas or liquid inlet 115 and detectoroutlet 113; fraction collector 14 in fluid communication with fractioncollector outlet 114 of shuttle valve 151; first detector 131 in fluidcommunication with detector outlet 113 of shuttle valve 151; fluidsupply 152 providing fluid to gas or liquid inlet 115 of shuttle valve151; and second detector 161 in fluid communication with detector outlet113 of shuttle valve 151.

As shown in FIG. 7, exemplary liquid chromatography system 70 compriseschromatography column 11; first shuttle valve 151 having chromatographycartridge inlet 111, fraction collector outlet 114, gas or liquid inlet115 and detector outlet 113; first detector 131 in fluid communicationwith detector outlet 113 of shuttle valve 151; fluid supply 152providing fluid to gas or liquid inlet 115 of shuttle valve 151; secondshuttle valve 171 having chromatography cartridge inlet 121, fractioncollector outlet 124, gas or liquid inlet 125 and detector outlet 123;second detector 161 in fluid communication with detector outlet 123 ofshuttle valve 171; fluid supply 172 providing fluid to gas or liquidinlet 125 of shuttle valve 171; and fraction collector 14 in fluidcommunication with fraction collector outlet 124 of shuttle valve 171.

In these exemplary embodiments, namely, exemplary liquid chromatographysystems 50 and 70, a method of analyzing a sample may further comprise astep of actively controlling fluid flow to second detector 16 (or seconddetector 161) via second splitter pump 17 (or second shuttle valve 171),as well as actively controlling fluid flow to first detector 13 (orfirst detector 131) via first splitter pump 15 (or first shuttle valve151). Although not shown in FIG. 5, it should be understood that firstsplitter pump 15 and/or second splitter pump 17 may be positioned withinexemplary liquid chromatography system 50 so as to push or pull fluidthrough first detector 13 and second detector 16 respectively.

In some exemplary embodiments, one or more optical absorbance detectors,such as one or more UV detectors, may be used to observe detectorresponses and changes in detector responses at one or more wavelengthsacross the absorbance spectrum. In these embodiments, one or more lightsources may be used in combination with multiple sensors within a singledetector or multiple detectors to detect light absorbance by a sample atmultiple wavelengths. For example, one or more UV detectors may be usedto observe detector responses and changes in detector responses at oneor more wavelengths across the entire UV absorbance spectrum.

In one exemplary method of analyzing a sample, the method comprises thestep of using an optical absorbance detector, such as an UV detector,comprising n sensors to observe a sample at n specific wavelengthsacross the entire UV absorbance spectrum; and collecting a new samplefraction in response to (i) a change in any one of the n detectorresponses at the n specific UV wavelengths, or (ii) a change in acomposite response represented by the n detector responses. The nsensors and multiple detectors, when present, may be positioned relativeto one another as desired to affect signal timing to a fractioncollector and/or another system component (e.g., another UV detector).

When utilizing whole-spectrum UV (or other spectrum range) analysis, thespectrum may be divided into any desired number of ranges of interest(e.g., every 5 nm range from 200 nm to 400 nm). Any significant changeover time in each spectrum range may be monitored. A sudden drop inreceived light energy (e.g., a drop in both the first and secondderivative of the detector response) within a given range may indicatethe arrival of a substance that absorbs light in the given wavelengthrange of interest. In this exemplary embodiment, the width of each rangecan be made smaller to increase precision; alternatively, the width ofeach range can be made larger so as to reduce the burden of calculation(i.e., fewer calculations per second, less memory required).

In other exemplary embodiments, a plurality of different types ofdetectors may be used to observe a variety of detector responses andchanges in the detector responses within a given system. In exemplaryliquid chromatography system 80 shown in FIG. 8, an evaporative particledetector (EPD), such as an evaporative light scattering detector (ELSD)(i.e., first detector 13) is used alone or in combination with an UVdetector (i.e., second detector 16). Exemplary liquid chromatographysystem 80 further comprises chromatography column 11; tee 12 havingfirst inlet 21, first outlet 22 and second outlet 23; fraction collector14; EPD 13 in fluid communication with second outlet 23 of tee 12;splitter pump 15 actively controlling fluid flow to EPD 13; and UVdetector 16 in fluid communication with first outlet 22 of tee 12. Inthis exemplary embodiment, the use of evaporative particle detectionoffers several advantages. Non-chromaphoric mobile phases must be usedwith UV detection or the mobile phase's background absorbance wouldobliterate the sample signal. This precludes using solvents such astoluene, pyridine and others that have otherwise valuablechromatographic properties. With evaporative particle detection, themobile phase chromaphoric properties are immaterial. As long as themobile phase is more volatile than the sample, it may be used withevaporative particle detection. This opens the opportunity to improveseparations through the use of highly selective chromaphoric solvents asthe mobile phase. Moreover, UV detectors will not detectnon-chromaphoric sample components. Fractions collected based on UVdetection only may contain one or more unidentifiable non-chromaphoriccomponents, which compromises fraction purity. Conversely,non-chromaphoric samples may be completely missed by UV detection andeither sent directly to waste or collected in fractions assumed to besample-free (blank fractions). The net result is lost productivity,contaminated fractions, or loss of valuable sample components. When anEPD (e.g., ELSD) is utilized alone or with UV detection in the flashsystem, chromaphoric and non-chromaphoric components are detected andcollected, improving fraction purity. Because a flash system thatincludes UV detector alone may miss sample components or incorrectlyflag pure fractions, many flash users will screen collected fractions bythin layer chromatography to confirm purity and confirm blank fractionsare truly blank. This is a time-consuming post-separation procedure thatslows down workflow. Those fractions discovered to contain more than onecomponent will frequently require a second chromatography step toproperly segregate the components.

In exemplary liquid chromatography system 80, signals 31 and 61 fromdetector (e.g., ELSD) 13 and UV detector 16 respectively may be sent tofraction collector 14 to initiate some activity from fraction collector14 such as, for example, collection of a new sample fraction. In desiredembodiments, in response to one or more detector signals 31 and 61 from(i) detector ELSD 13, (ii) UV detector 16, or (iii) both ELSD 13 and UVdetector 16, fraction collector 14 collects a new sample fraction.

Similar to exemplary liquid chromatography system 80, in exemplaryliquid chromatography system 60 shown in FIG. 6, signals 311 and 611from ELSD 131 and UV detector 161 respectively may be sent to fractioncollector 14 to initiate some activity from fraction collector 14 suchas, for example, collection of a new sample fraction. In desiredembodiments, in response to one or more detector signals 311 and 611from (i) ELSD 131, (ii) UV detector 161, or (iii) both ELSD 131 and UVdetector 161, fraction collector 14 collects a new sample fraction.

As discussed above, UV detector 16 (or UV detector 161) may comprise nsensors operatively adapted to observe a sample at n specificwavelengths across a portion of or the entire UV absorbance spectrum. Inexemplary liquid chromatography system 80 shown in FIG. 8, in responseto (i) a single signal from either one of ELSD 13 or UV detector 16,(ii) two or more signals from both ELSD 13 and UV detector 16, or (iii)a composite signal comprising two or more detector responses (i.e., upto n detector responses) at the two or more specific UV wavelengths(i.e., up to n specific UV wavelengths), fraction collector 14 collectsa new sample fraction. Similarly, in exemplary liquid chromatographysystem 60 shown in FIG. 6, in response to (i) a single signal fromeither one of ELSD 131 or UV detector 161, (ii) two or more signals fromboth ELSD 131 and UV detector 161, or (iii) a composite signalcomprising two or more detector responses (i.e., up to n detectorresponses) at the two or more specific UV wavelengths (i.e., up to nspecific UV wavelengths), fraction collector 14 collects a new samplefraction.

Further, in exemplary liquid chromatography system 80, UV detector 16may be used to produce a detector signal (not shown) that (1) results(i) from a single detector response from a single sensor or (ii) from ndetector responses of n sensors with n being greater than 1, and (2) issent to at least one of splitter pump 15, ELSD 13 and tee 12. Inaddition, a detector signal (not shown) resulting from a detectorresponse in ELSD 13 may be sent to UV detector 16 to change one or moresettings of UV detector 16. Similarly, in exemplary liquidchromatography system 60 shown in FIG. 6, UV detector 161 may be used toproduce a detector signal (not shown) that (1) results (i) from a singledetector response from a single sensor or (ii) from n detector responsesof n sensors with n being greater than 1, and (2) is sent to at leastone of shuttle valve 151 and ELSD 13. In addition, a detector signal(not shown) resulting from a detector response in ELSD 131 may be sentto UV detector 161 to change one or more settings of UV detector 161.

As shown in exemplary liquid chromatography system 90 shown in FIG. 9,the position of different types of detectors within a given system maybe adjusted as desired to provide one or more system process features.In exemplary liquid chromatography system 90, ELSD 13 is positioneddownstream from UV detector 16. In such a configuration, UV detector 16is positioned to be able to provide a detector response and generatesignal 61 (e.g., a signal that results (i) from a single detectorresponse from a single sensor or (ii) from n detector responses of nsensors with n being greater than 1) for fraction collector 14 prior tothe generation of signal 31 from ELSD 13. UV detector 16 is alsopositioned to be able to provide a detector response and generate asignal (not shown) (e.g., a signal that results (i) from a singledetector response from a single sensor or (ii) from n detector responsesof n sensors with n being greater than 1) for at least one of splitterpump 15, ELSD 13 and tee 12 so as to activate or deactivate splitterpump 15, ELSD 13 and/or tee 12.

Although not shown, it should be understood that a shuttle valve may beused in place of tee 12 and splitter pump 15 within exemplary liquidchromatography system 90 shown in FIG. 9 to provide similar systemprocess features. In such a configuration, UV detector 16 is positionedto be able to provide a detector response and generate signal 61 (e.g.,a signal that results (i) from a single detector response from a singlesensor or (ii) from n detector responses of n sensors with n beinggreater than 1) for fraction collector 14 prior to the generation ofsignal 31 from ELSD 13. UV detector 16 is also positioned to be able toprovide a detector response and generate a signal (not shown) (e.g., asignal that results (i) from a single detector response from a singlesensor or (ii) from n detector responses of n sensors with n beinggreater than 1) for at least one of a shuttle valve and ELSD 13 so as toactivate or deactivate the shuttle valve and/or ELSD 13. Even thoughsystems 60, 80, and 90 refer to ELSD and UV as the detectors, anydestructive detector, such as EPD, may be utilized for the ELSD, and anynon-destructive detector may be utilized in place of the UV detector.

In other exemplary embodiments, the liquid chromatography system of thepresent invention may comprise a non-destructive system comprising twoor more non-destructive detectors (e.g., one or more optical absorbancedetectors, such as the UV detectors described above) with no destructivedetectors (e.g., a mass spectrometer) present in the system. In oneexemplary embodiment, the liquid chromatography system comprises twooptical absorbance detectors such as UV detectors, and the method ofanalyzing a sample comprises the step of using two or more detectors toobserve a sample at two or more specific wavelengths; and collecting anew sample fraction in response to (i) a change in a first detectorresponse at a first wavelength, (ii) a change in a second detectorresponse at a second wavelength, or (iii) a change in a compositeresponse represented by the first detector response and the seconddetector response. In these embodiments, the first wavelength may besubstantially equal to or different from the second wavelength.

In embodiments utilizing two or more optical absorbance detectors, suchas two or more UV detectors, the optical absorbance detectors may bepositioned within a given liquid chromatography system so as to provideone or more system advantages. The two or more optical absorbancedetectors may be positioned in a parallel relationship with one anotherso that a sample reaches each detector at substantially the same time,and the two or more optical absorbance detectors may produce and sendsignals (i.e., from first detector and second detector responses) atsubstantially the same time to a fraction collector.

In a further embodiment, a non-destructive detector (e.g., RI detector,UV detector, etc.) may be used alone or in combined with a destructivedetector (e.g., EPD, mass spectrometer, spectrophotometer, emissionspectroscopy, NMR, etc.). For example, a destructive detector, such as amass spectrometer detector, enables simultaneous detection of thecomponent peak and chemical entity associated with the peak. This allowsfor immediate determination of the fraction that contains the targetcompound. With the other detection techniques, post separationdetermination of which fraction contains the target compound may berequired, such as by, for example, spectrophotometry, mass spectrometry,emission spectroscopy, NMR, etc. If two or more chemical entities eluteat the same time from the flash cartridge (i.e., have the same retentiontime), they will be deposited in the same vial by the system when usingcertain detectors (i.e., those detectors that cannot identifydifferences between the chemical entities) because these detectorscannot determine chemical composition. In an exemplary embodiment wherea mass spectrometer detector is utilized as the destructive detector,all compounds that elute at the same time may be identified. Thiseliminates the need to confirm purity after separation.

In any of the above-described liquid chromatography systems, it may beadvantageous to position at least one detector, such as at least one UVdetector, downstream from (e.g., in series with) at least one otherdetector, such as at least one other UV detector or an ELSD. In such anembodiment, a first detector response in a first detector can be used toproduce and send a signal to at least one of (1) a splitter pump, (2) ashuttle valve, (3) a second detector and (4) a tee. For example, a firstdetector response in a first detector can be used to produce and send asignal to a splitter pump or a shuttle valve to (i) activate thesplitter pump or the shuttle valve, (ii) deactivate the splitter pump orthe shuttle valve, (iii) change one or more flow or pressure settings ofthe splitter pump or the shuttle valve, or (iv) any combination of (i)to (iii). Suitable flow and pressure settings include, but are notlimited to, the flow and pressure settings described above. Typically,the signal is in the form of, for example, an electrical signal, apneumatic signal, a digital signal, or a wireless signal.

In some embodiments, multiple detectors (i.e., two or more detectors)may be positioned so that each detector can send a signal to at leastone of (1) a splitter pump, (2) a shuttle valve, (3) another detectorand (4) a tee independently of the other detectors in the system. Forexample, multiple optical absorbance detectors (e.g., UV detectors) maybe positioned within a given system to provide independent signals to ashuttle valve to cause the shuttle valve to provide actively controlledfluid sampling to another detector such as an ELSD.

In other embodiments, a first detector response in a first detector canbe used to produce and send a signal to a second detector to (i)activate the second detector, (ii) activate the second detector at awavelength substantially similar to a first wavelength used in the firstdetector, (iii) activate the second detector at a wavelength other thanthe first wavelength used in the first detector, (iv) deactivate thesecond detector, (v) change some other setting of the second detector(e.g., the observed wavelength of the second detector), or (vi) anycombination of (i) to (v).

In yet other embodiments, a first detector response in a first detectorcan be used to produce and send a signal to a tee to (i) open a valve or(ii) close a valve so as to start or stop fluid flow through a portionof the liquid chromatography system. As discussed above, typically, thesignal is in the form of, for example, an electrical signal, a pneumaticsignal, a digital signal, or a wireless signal.

C. Generation of a Signal from a Detector Response

The methods of the present invention may further comprise the step ofgenerating a signal from one or more detector responses. In someembodiments, such as exemplary liquid chromatography system 10 shown inFIG. 1, a single detector detects the presence of a sample component andproduces a detector response based on the presence and concentration ofa sample component within a fluid stream. In other embodiments, such asexemplary liquid chromatography system 50 shown in FIG. 6, two or moredetectors may be used to detect the presence of one or more samplecomponents, and produce two or more detector responses based on thepresence and concentration of one or more sample components within afluid stream.

As discussed above, a given detector provides one or more detectorresponses that may be used to generate and send a signal to one or morecomponents (e.g., a fraction collector, another detector, a splitterpump, a shuttle valve, or a tee) within a liquid chromatography systemas described herein. Typically, a change in a given detector responsetriggers the generation and sending of a signal. Changes in a givendetector response that might trigger the generation and sending of asignal to one or more components include, but are not limited to, achange in a detector response value, reaching or exceeding a thresholddetector response value, a slope of the detector response value overtime, a threshold slope of the detector response value over time, achange in a slope of the detector response value over time, a thresholdchange in a slope of the detector response value over time, or anycombination thereof.

In one exemplary embodiment, the methods of the present inventioncomprise the step of generating a detector signal from at least onedetector, the detector signal being generated in response to (i) theslope of a detector response as a function of time (i.e., the firstderivative of a detector response), (ii) a change in the slope of thedetector response as a function of time (i.e., the second derivative ofthe detector response), (iii) optionally, a threshold detector responsevalue, or (iv) any combination of (i) to (iii) with desired combinationscomprising at least (i) or at least (ii). In this exemplary embodiment,a substance is recognized from the shape of the detector response,specifically the first and/or second derivative of the detector responseover time (i.e., slope and change in slope, respectively). Inparticular, a computer program analyzes the time sequence of detectorresponse values and measures its rate of change (i.e., the firstderivative), and the rate of the rate of change (i.e., the secondderivative). When both the first derivative and the second derivativeare increasing, a substance is beginning to be detected. Similarly, whenboth the first derivative and the second derivative are decreasing, thesubstance is ceasing to be detected.

Real-world detector values are typically noisy (e.g., jagged), so it isdesirable to utilize low-pass numerical filtering (e.g., smoothing) overtime. Consequently, the step of generating a detector signal from atleast one detector desirably further comprises low-pass numericalfiltering of (i) slope data over time, (ii) change in slope data overtime, (iii) optionally, a threshold detector response value, or (iv) anycombination of (i) to (iii) to distinguish actual changes in (i) slopedata over time, (ii) change in slope data over time, (iii) optionally, athreshold detector response value, or (iv) any combination of (i) to(iii) from possible noise in the detector response. In desiredembodiments, a finite impulse response (FIR) filter or infinite impulseresponse (IIR) filter may be utilized for low-pass numerical filteringof data over time (e.g., perhaps just an average of several samples).Typically, the decision algorithm utilizes a small number of sequentialsuccesses in time as confirmation of a real detector response/signal,and not noise.

In other embodiments, the method of analyzing a sample may comprisegenerating a composite signal comprising a detection response componentfrom each detector, and collecting a new sample fraction in response toa change in the composite signal. In these embodiments, the step ofgenerating a composite signal may comprise mathematically correlating(i) a detector response value, (ii) the slope of a given detectorresponse as a function of time (i.e., the first derivative of a givendetector response), (iii) a change in the slope of the given detectorresponse as a function of time (i.e., the second derivative of the givendetector response), or (iv) any combination of (i) to (iii) from eachdetector (i.e., each of the two or more detectors). For example, in someembodiments, the composite signal may comprise (i) the product ofdetector response values for each detector (i.e., each of two or moredetectors) at a given time, (ii) the product of the first derivatives ofthe detector responses at a given time, (iii) the product of the secondderivatives of the detector responses at a given time, or (iv) anycombination of (i) to (iii).

In other embodiments in which a composite signal is used, the step ofgenerating a composite signal may comprise mathematically correlating(i) a detector response value, (ii) the slope of a given detectorresponse as a function of time (i.e., the first derivative of a givendetector response), (iii) a change in the slope of the given detectorresponse as a function of time (i.e., the second derivative of the givendetector response), or (iv) any combination of (i) to (iii) from eachsensor within a detector (i.e., n sensors observing a sample at nspecific wavelengths) alone or in combination with any other detectorresponses present in the system. For example, in some embodiments, thecomposite signal may comprise (i) the product of detector responsevalues for each sensor within a detector (i.e., n sensors observing asample at n specific wavelengths) and any additional detector responsevalues from other detectors (e.g., from an ELSD used in combination withan UV detector) at a given time, (ii) the product of the firstderivatives of the detector responses for each sensor within a detector(i.e., n sensors observing a sample at n specific wavelengths) and anyadditional detector responses from other detectors at a given time,(iii) the product of the second derivatives of the detector responsesfor each sensor within a detector (i.e., n sensors observing a sample atn specific wavelengths) and any additional detector responses from otherdetectors at a given time, or (iv) any combination of (i) to (iii).

D. Collection of One or More Sample Fractions

The methods of the present invention may further comprise using afraction collector, such as exemplary fraction collector 14 shown inFIGS. 1-3A and 4-9, to collect one or more sample fractions in responseto one or more signals from at least one detector in a given liquidchromatography system. For example, in exemplary liquid chromatographysystems 10, 20 and 30 shown in FIGS. 1, 2 and 3A respectively, methodsof analyzing a sample may further comprise the step of collecting one ormore sample fractions in response to one or more signals from firstdetector 13. In exemplary liquid chromatography systems 40, 50 and 60shown in FIGS. 4, 5 and 6 respectively, methods of analyzing a samplemay further comprise the step of collecting one or more sample fractionsin response to one or more signals from first detector 13 (or firstdetector 131), second detector 16 (or second detector 161), or bothfirst and second detectors 13 and 16 (or both first and second detectors131 and 161).

In some embodiments of the present invention, the fraction collector isoperatively adapted to recognize, receive and process one or moresignals from at least one detector, and collect one or more samplefractions based on the one or more signals. In other embodiments,additional computer or microprocessing equipment is utilized to processone or more signals from at least one detector and subsequently provideto the fraction collector a recognizable signal that instructs thefraction collector to collect one or more sample fractions based on oneor more signals from the additional computer or microprocessingequipment.

As discussed above, system components may be positioned within a givenliquid chromatography system to provide one or more system properties.For example, at least one detector may be positioned within a givenliquid chromatography system so as to minimize any time delay between(i) the detection of a given detector response and (ii) the step ofcollecting a sample fraction based on a signal generated from thedetector response. In exemplary embodiments of the present invention,the liquid chromatography system desirably exhibits a maximum time delayof a given detector signal to the fraction collector (i.e., the timedelay between (i) the detection of a given detector response and (ii)the step of collecting a sample fraction based on a signal generatedfrom the detector response) of less than about 2.0 seconds (s) (or lessthan about 1.5 s, or less than about 1.0 s, or less than about 0.5 s).

In exemplary embodiments of the present invention utilizing two or moredetectors or at least one detector comprising n sensors (as describedabove), the liquid chromatography system desirably exhibits a maximumtime delay for any detector signal from any detector to the fractioncollector (i.e., the time delay between (i) the detection of a givendetector response and (ii) the step of collecting a sample fractionbased on a signal (e.g., single or composite signal) generated from thedetector response) of less than about 2.0 s (or less than about 1.5 s,or less than about 1.0 s, or less than about 0.5 s).

E. Sample Component(s) Separation Step

The methods of the present invention utilize a liquid chromatography(LC) step to separate compounds within a given sample. Depending on theparticular sample, various LC columns, mobile phases, and other processstep conditions (e.g., feed rate, gradient, etc.) may be used.

A number of LC columns may be used in the present invention. In general,any polymer or inorganic based normal phase, reversed phase, ionexchange, affinity, hydrophobic interaction, hydrophilic interaction,mixed mode and size exclusion columns may be used in the presentinvention. Exemplary commercially available columns include, but are notlimited to, columns available from Grace Davison Discovery Sciencesunder the trade names VYDAC®, GRACERESOLV™, DAVISIL®, ALLTIMA™, VISION™,GRACEPURE™, EVEREST®, and DENALI®, as well as other similar companies.

A number of mobile phase components may be used in the presentinvention. Suitable mobile phase components include, but are not limitedto, acetonitrile, dichloromethane, ethyl acetate, heptane, acetone,ethyl ether, tetrahydrofuran, chloroform, hexane, methanol, isopropylalcohol, water, ethanol, buffers, and combinations thereof.

F. User Interface Steps

The methods of analyzing a sample in the present invention may furthercomprise one or more steps in which an operator or user interfaces withone or more system components of a liquid chromatography system. Forexample, the methods of analyzing a sample may comprise one or more ofthe following steps: inputting a sample into the liquid chromatographysystem for testing; adjusting one or more settings (e.g., flow orpressure settings, wavelengths, etc.) of one or more components withinthe system; programming at least one detector to generate a signal basedon a desired mathematical algorithm that takes into account one or moredetector responses from one or more sensors and/or detectors;programming one or more system components (other than a detector) togenerate a signal based on a desired mathematical algorithm that takesinto account one or more detector responses; programming a fractioncollector to recognize a signal (e.g., a single or composite signal)from at least one detector, and collect one or more sample fractionsbased on a received signal; programming one or more system components(other than a fraction collector) to recognize an incoming signal fromat least one detector, convert the incoming signal into a signalrecognizable and processable by a fraction collector so that thefraction collector is able to collect one or more sample fractions basedon input from the one or more system components; and activating ordeactivating one or more system components (e.g., a tee valve, asplitter pump, a shuttle valve or a detector) at a desired time or inresponse to some other activity within the liquid chromatography system(e.g., a detector response displayed to the operator or user).

II. Apparatus for Analyzing Samples

The present invention is also directed to an apparatus and apparatuscomponents capable of analyzing a sample or capable of contributing tothe analysis of a sample using one or more of the above-described methodsteps.

As described above, in some exemplary embodiments of the presentinvention, an apparatus for analyzing a sample may comprise (i) achromatography column; (ii) a tee having a first inlet, a first outletand a second outlet; (iii) a fraction collector in fluid communicationwith the first outlet of the tee; (iv) a first detector in fluidcommunication with the second outlet of the tee; and (v) a splitter pumppositioned in fluid communication with the second outlet of the tee andthe first detector with the splitter pump being operatively adapted toactively control fluid flow to the first detector. In other exemplaryembodiments of the present invention, a shuttle valve may be used inplace of a tee/splitter pump combination to actively control fluid flowto the first detector.

Although not shown in FIGS. 1-9, any of the above-described apparatus(e.g., exemplary liquid chromatography systems 10 to 90) or apparatuscomponents may further comprise system hardware that enables (i) therecognition of a detector response value or a change in a detectorresponse value, (ii) the generation of a single from the detectorresponse value or a change in a detector response value, (iii) thesending of a signal to one or more system components, (iv) therecognition of a generated signal by a receiving component, (v)processing of the recognized signal within the receiving component, and(vi) the initiation of a process step of the receiving component inresponse to the recognized signal.

In one embodiments, the apparatus (e.g., exemplary liquid chromatographysystems 10 to 90) or a given apparatus component may further comprisesystem hardware that enables a first detector to send an activationsignal to a splitter pump or a shuttle valve to (i) activate thesplitter pump or shuttle valve, (ii) deactivate the splitter pump orshuttle valve, (iii) change one or more flow or pressure settings of thesplitter pump or shuttle valve, or (iv) any combination of (i) to (iii).Suitable flow and pressure settings may include, but are not limited to,(i) a valve position, (ii) splitter pump or shuttle valve pressure,(iii) air pressure to a valve, or (iv) any combination of (i) to (iii).

In some embodiments, a splitter pump may be positioned between a tee anda first detector (see, for example, spatter pump 15 positioned betweentee 12 and first detector 13 in FIG. 1). In other embodiments, a firstdetector may be positioned between a tee and the splitter pump (see, forexample, first detector 13 positioned between tee 12 and splitter pump15 in FIG. 2).

In other exemplary embodiments, the apparatus of the present inventioncomprise (i) a chromatography column; (ii) two or more detectors; and(iii) a fraction collector in fluid communication with the two or moredetectors with the fraction collector being operatively adapted tocollect one or more sample fractions in response to one or more detectorsignals from the two or more detectors. In some embodiments, the two ormore detectors comprise two or more non-destructive detectors (e.g., twoor more UV detectors) with no destructive detectors (e.g., massspectrometer) in the system.

When two or more detectors are present, a splitter pump or shuttle valvemay be used to split a volume of fluid flow between a first detector anda second detector. In other embodiments, a splitter pump or shuttlevalve may be used to initiate or stop fluid flow to one detector inresponse to a detector response from another detector. In addition,multiple splitter pumps and/or shuttle valves may be used in a givensystem to actively control fluid flow to two or more detectors.

As discussed above, the apparatus may further comprise system hardwarethat enables generation of a detector signal from one or more detectorresponses. In one exemplary embodiment, the apparatus comprises systemhardware that enables generation of a detector signal that is generatedin response to (i) the slope of a detector response as a function oftime (i.e., the first derivative of a detector response), (ii) a changein the slope of the detector response as a function of time (i.e., thesecond derivative of the detector response), (iii) optionally, athreshold detector response value, or (iv) any combination of (i) to(iii) with desired combinations comprising at least (i) or at least(ii). The system hardware desirably further comprises low-pass numericalfiltering capabilities for filtering (i) slope data, (ii) change inslope data, (iii) optionally, a threshold detector response value, or(iv) any combination of (i) to (iii) over time to distinguish actualchanges in (i) slope data, (ii) change in slope data, (iii) optionally,a threshold detector response value, or (iv) any combination of (i) to(iii) from possible noise in a given detector response.

In multi-detector systems, system hardware may also be used to enablethe generation of a composite signal comprising a detection responsecomponent from each detector, as well as detection response componentsfrom multiple sensors within a given detector. In these embodiments, thesystem hardware is operatively adapted to send a command/signal to afraction collector instructing the fraction collector to collect a newsample fraction in response to a change in the composite signal. Thecomposite signal may comprise a mathematical correlation between (i) adetector response value, (ii) the slope of a given detector response asa function of time (i.e., the first derivative of a given detectorresponse), (iii) a change in the slope of the given detector response asa function of time (i.e., the second derivative of the given detectorresponse), or (iv) any combination of (i) to (iii) from each detector.For example, the composite signal may comprise (i) the product ofdetector response values for each detector at a given time, (ii) theproduct of the first derivatives of the detector responses at a giventime, (iii) the product of the second derivatives of the detectorresponses at a given time, or (iv) any combination of (i) to (iii).

In one desired configuration, the apparatus for analyzing a samplecomprising at least one detector operatively adapted to observe a sampleat two or more specific optical wavelengths (e.g., within the UVspectrum), and system hardware that enables a fraction collector tocollect a new sample fraction in response to (i) a change in a detectorresponse at a first wavelength, (ii) a change in a detector response ata second wavelength, or (iii) a change in a composite responserepresented by detector responses at the first and second wavelengths.Each detector can operate at the same wavelength(s), at differentwavelengths, or multiple wavelengths. Further, each detector may be in aparallel relationship with one another, in series with one another, orsome combination of parallel and series detectors.

As discussed above, in one exemplary embodiment, the apparatus maycomprise a single detector comprising n sensors operatively adapted toobserve a sample at n specific optical wavelengths across a portion ofor the entire UV absorbance spectrum (or any other portion of theabsorbance spectrum using some other type of detector), and systemhardware that enables a fraction collector to collect a new samplefraction in response to (i) a change in any one of the n detectorresponses at the n specific optical wavelengths, or (ii) a change in acomposite response represented by the n detector responses.

When a splitter pump or shuttle valve is present to actively controlfluid flow to at least one detector, the apparatus for analyzing asample may further comprise system hardware that enables generation ofan activation signal to the splitter pump or shuttle valve to (i)activate the splitter pump or shuttle valve, (ii) deactivate thesplitter pump or shuttle valve, (iii) change one or more flow orpressure settings of the splitter pump or shuttle valve, or (iv) anycombination of (i) to (iii). The activation signal may be generated, forexample, by a system operator or by a system component, such as adetector (i.e., the activation signal being generated and sent by thedetector in response to a detector response value or change in adetector response value of the detector as discussed above).

In an even further embodiment according to the present invention, anapparatus for analyzing a sample of fluid using chromatography includesa first fluid path of effluent from a chromatography column orcartridge; at least one detector that is capable of analyzing the sampleof fluid; and a shuttle valve that transfers an aliquot sample of fluidfrom the first fluid path to the detector(s) without substantiallyaffecting the flow properties of fluid through the first fluid path. Theflow of the fluid through the first fluid path may be substantiallylaminar, due to the first fluid path or channel being substantiallylinear or straight through at least a portion of the valve. In a furtherexemplary embodiment, the pressure of the fluid through the first fluidpath remains substantially constant and/or it does not substantiallyincrease. In another embodiment, the flow rate of the fluid may besubstantially constant through the first fluid path. In an alternativeembodiment, a second fluid path is utilized to carry the aliquot sampleof fluid from the shuttle valve to the detector(s). The flow of fluidthrough the second fluid path may be substantially laminar due to thesecond fluid path or channel being substantially linear or straightthrough at least a portion of the valve. In an exemplary embodiment, thepressure of fluid through the second fluid path is substantiallyconstant and/or it does not substantially increase. In furtherembodiment, the flow rate of fluid may be substantially constant throughthe second fluid path.

In an even further exemplary embodiment, an apparatus for analyzing asample of fluid using chromatography includes a first fluid path ofeffluent from a chromatography column; a second fluid path that carriesthe sample of fluid to at least one detector that is capable ofanalyzing the sample; and a shuttle valve that transfers an aliquotsample of fluid from the first fluid path to the second fluid path whilemaintaining a continuous second fluid path through the shuttle valve. Inone embodiment, a continuous first flow path through the shuttle valveis maintained when the aliquot sample of fluid is removed from the firstfluid path. In another embodiment, continuous first and second flowpaths through the shuttle valve are maintained when the aliquot sampleof fluid is removed from the first fluid path and transferred to thesecond fluid path.

In exemplary embodiments of the present invention, the apparatus foranalyzing a sample further comprises a fraction collector that isoperatively adapted to collect one or more sample fractions in responseto one or more detector signals from (i) a first detector, (ii) a seconddetector (or any number of additional detectors), or (iii) both thefirst and second detectors (or any number of additional detectors). Whenmultiple detectors are utilized, the apparatus may comprise a fractioncollector operatively adapted to collect a new sample fraction inresponse to a change in a composite signal that accounts for one or moredetector responses from each detector as described above.

As discussed above, in some exemplary embodiments, the apparatus foranalyzing a sample comprises a fraction collector that is operativelyadapted to recognize, receive and process one or more signals from atleast one detector, and collect one or more sample fractions based onthe one or more signals. In other embodiments, the apparatus foranalyzing a sample comprises additional computer or microprocessingequipment that is capable of processing one or more signals from atleast one detector and converting an incoming signal into a signal thatis recognizable by the fraction collector. In this later embodiment, thefraction collector collects one or more sample fractions based on theone or more signals from the additional computer or microprocessingequipment, not from signal processing components of the fractioncollector.

It should be noted that any of the above-described exemplary liquidchromatography systems may comprise any number of detectors, splitterpumps, tees, and shuttle valves, which may be strategically placedwithin a given system to provide one or more system properties. Forexample, although not shown in exemplary liquid chromatography system 60in FIG. 6, an additional detector could be positioned between column 11and shuttle valve 151 and/or between shuttle valve 151 and detector 161.Although not shown in exemplary liquid chromatography system 70 in FIG.7, an additional detector could be positioned between column 11 andshuttle valve 151 and/or between shuttle valve 151 and shuttle valve 171and/or between shuttle valve 171 and fraction collector 14. Additionaldetectors may be similarly positioned within exemplary liquidchromatography systems 80 and 90 shown in FIGS. 8 and 9 respectively,

A number of commercially available components may be used in theapparatus of the present invention as discussed below.

A. Chromatography Columns

Any known chromatography column may be used in the apparatus of thepresent invention. Suitable commercially available chromatographycolumns include, but are not limited to, chromatography columnsavailable from Grace Davison Discovery Sciences (Deerfield, Ill.) underthe trade designations GRACEPURE™, GRACERESOLV™, VYDAC® and DAVISIL®.

B. Detectors

Any known detector may be used in the apparatus of the presentinvention. Suitable commercially available detectors include, but arenot limited to, UV detectors available from Ocean Optics (Dunedin, Fla.)under the trade designation USB 2000™; evaporative light scatteringdetectors (ELSDs) available from Grace Davison Discovery Sciences(Deerfield, Ill.) under the trade designation 3300 ELSD™; massspectrometers (MSs) available from Waters Corporation (Milford, Mass.)under the trade designation ZQ™; condensation nucleation lightscattering detectors (CNLSDs) available from Quant (Blaine, Minn.) underthe trade designation QT-500™; corona discharge detectors (CDDs)available from ESA (Chelmsford, Mass.) under the trade designationCORONA CAD™; refractive index detectors (RIDS) available from WatersCorporation (Milford Mass.) under the trade designation 2414; andfluorescence detectors (FDs) available from Laballiance (St. Collect,Pa.) under the trade designation ULTRAFLOR™.

In some embodiments, a commercially available detector may need to bemodified or programmed or a specific detector may need to be built inorder to perform one or more of the above-described method steps of thepresent invention.

C. Splitter Pumps

Any known splitter pump may be used in the apparatus of the presentinvention. Suitable commercially available splitter pumps include, butare not limited to, splitter pumps available from KNF (Trenton, N.J.)under the trade designation LIQUID MICRO™.

D. Shuttle Valves

Any known shuttle valve may be used in the apparatus of the presentinvention. Suitable commercially available shuttle valves include, butare not limited to, shuttle valves available from Valco (Houston, Tex.)under the trade designation CHEMINERT™, Rheodyne® shuttle valveavailable from Hex Corporation under the trade name MRA® and acontinuous flow shuttle valve as described herein.

E. Fraction Collectors

Any known fraction collector may be used in the apparatus of the presentinvention. Suitable commercially available fraction collectors include,but are not limited to, fraction collectors available from Gilson(Middleton, Wis.) under the trade designation 215.

In some embodiments, a commercially available fraction collector mayneed to be modified and/or programmed or a specific fraction collectormay need to be built in order to perform one or more of theabove-described method steps of the present invention. For example,fraction collectors that are operatively adapted to recognize, receiveand process one or more signals from at least one detector, and collectone or more sample fractions based on the one or more signals are notcommercially available at this time.

III. Computer Software

The present invention is further directed to a computer readable mediumhaving stored thereon computer-executable instructions for performingone or more of the above-described method steps. For example, thecomputer readable medium may have stored thereon computer-executableinstructions for: adjusting one or more settings (e.g., flow settings,wavelengths, etc.) of one or more components within the system;generating a signal based on a desired mathematical algorithm that takesinto account one or more detector responses; recognizing a signal fromat least one detector; collecting one or more sample fractions based ona received signal; recognizing an incoming signal from at least onedetector, convert the incoming signal into a signal recognizable andprocessable by a fraction collector so that the fraction collector isable to collect one or more sample fractions based on input from the oneor more system components; and activating or deactivating one or moresystem components (e.g., a tee valve, a splitter pump, a shuttle valve,or a detector) at a desired time or in response to some other activitywithin the liquid chromatography system (e.g., a detector response).

IV. Applications/Uses

The above-described methods, apparatus and computer software may be usedto detect the presence of one or more compounds in a variety of samples.The above-described methods, apparatus and computer software findapplicability in any industry that utilizes liquid chromatographyincluding, but not limited to, the petroleum industry, thepharmaceutical industry, analytical labs, etc.

EXAMPLES

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

Example 1

In this example, the flash REVELEFIS™ system (available from GraceDavison Discovery Sciences) was utilized, 4 mL of a mixture containingsucrose and aspirin was injected into a 4 g GRACERESOLV™ C18 flashcolumn (available from Grace Davison Discovery Sciences), which wasmounted in the flash system. A 50/50 methanol/water mobile phase waspumped through the system using an ALLTECH® model 300 LC pump. Thecolumn effluent was directed to a KNF splitter pump that diverted 300uL/min of the column effluent to an ALLTECH® 3300 ELSD. The balance ofthe effluent flowed through an Ocean Optics UV detector to a Gilsonfraction collector.

The sucrose and aspirin were separated on the flash column. Both thesucrose and the aspirin were detected by the ELSD. The UV detector onlydetected the aspirin. Both detectors responded to the aspirin at thesame time. The fraction collector deposited the sucrose and aspirin inseparate collection vials in response to a composite signal from the UVand ELSD detectors.

Example 2

In this example, the flash REVELERIS™ system (available from GraceDavison Discovery Sciences) was utilized. 4 mL of a mixture containingdioctyl phthalate and butyl paraben was injected into a 4 g GraceResolv™C18 flash cartridge (available from Grace Davison Discovery Sciences),which was mounted in the flash system. A 80/20 methanol/water mobilephase was pumped through the system using an Alltech® model 300 LC pump.The column effluent was directed to a shuttle valve as described hereinthat diverted 300 uL/min of the column effluent to an Alltech® 3300ELSD. The balance of the effluent flowed through an Ocean Optics UVdetector to a Gilson fraction collector.

This two component mixture contains a non-chromaphoric compound (onethat does not absorb UV light) and a chromaphoric compound. Thenon-chromaphoric compound elutes from the flash cartridge first. FIG. 11depicts a chromatogram that illustrates only the ELSD identifies all thecomponents in the sample, as is evidenced by the two peaks on thechromatogram. The UV detector does not identify the non-chromaphoriccompound (identified as the first peak by the ELSD), even at twowavelengths. Only the ELSD signal will be able to properly control thefraction collector, capturing both compounds. If the UV detector drovethe fraction collector, (as would be the case in conventional Flashsystems), the first compound would be sent to waste or improperlydeposited in collection vessels without knowledge that these fractionscontained desired sample. In conventional flash instruments, allfractions are screened by thin layer chromatography (TLC) after thechromatographic separation to find compounds that the UV detector maynot have identified. This Example demonstrates that flash instrumentsequipped with an ELSD according to the present invention are able toidentify and separate both chromaphoric and non-chromaphoric compounds,and post-separation TLC screening is not required.

While the invention has been described with a limited number ofembodiments, these specific embodiments are not intended to limit thescope of the invention as otherwise described and claimed herein. It maybe evident to those of ordinary skill in the art upon review of theexemplary embodiments herein that further modifications, equivalents,and variations are possible. All parts and percentages in the examples,as well as in the remainder of the specification, are by weight unlessotherwise specified. Further, any range of numbers recited in thespecification or claims, such as that representing a particular set ofproperties, units of measure, conditions, physical states orpercentages, is intended to literally incorporate expressly herein byreference or otherwise, any number falling within such range, includingany subset of numbers within any range so recited. For example, whenevera numerical range with a lower limit, RL, and an upper limit RU, isdisclosed, any number R falling within the range is specificallydisclosed. In particular, the following numbers R within the range arespecifically disclosed: R=RL+k(RU−RL), where k is a variable rangingfrom 1% to 100% with a 1% increment, e.g., k is 1%, 2%, 3%, 4%, 5% . . .50%, 51%, 52% . . . 95%, 96%, 97%, 98%, 99%, or 100%. Moreover, anynumerical range represented by any two values of R, as calculated aboveis also specifically disclosed. Any modifications of the invention, inaddition to those shown and described herein, will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims. All publications cited herein are incorporated byreference in their entirety.

What is claimed is:
 1. A method of detecting and collecting one or moresample components within a first sample stream in a chromatographysystem comprising: separating one or more sample components from a fluidmixture during a chromatographic run using a chromatography column toform the first sample stream; generating a second sample streamcomprising a carrier gas; moving a portion of the first sample stream tothe second sample stream, which is in fluid communication with at leastone destructive detector; generating at least one signal during thechromatographic run from the destructive detector; and collecting theone or more components from the first sample stream in a fractioncollector in response to a change in the at least one signal.
 2. Amethod of claim 1, wherein said generating and collecting steps areperformed during the chromatographic run.
 3. The method of claim 1,including at least one destructive detector selected from the groupconsisting of evaporative light scattering detectors (ELSD), massspectrometers (MS), condensation nucleation light scattering detectors(CNLSD), and corona discharge detectors.
 4. The method of claim 1,wherein said chromatography system further comprises at least onenon-destructive detector selected from the group consisting of opticalabsorbance detectors, refractive index detectors (RID), fluorescencedetectors (FD), chiral detectors (CD), and conductivity detectors. 5.The method of claim 4, wherein the non-destructive detector comprises atleast one optical absorbance detector.
 6. The method of claim 5, whereinthe optical absorbance detector observes two or more optical wavelengthsso as to produce two or mare detector response values.
 7. The method ofclaim 1, wherein the destructive detector comprises an evaporative lightscattering detector.
 8. The method of claim 1 wherein said step ofmoving is performed with use of a splitter pump; a shuttle valve; or acombination of a splitter pump and a shuttle valve; wherein the splitterpump, the shuttle valve, or the combination are in fluid communicationwith the at least one destructive detector.
 9. The method of claim 1,wherein said step of actively moving comprises moving an aliquot fromfirst sample stream at a frequency of at least 1 aliquot every 10seconds.
 10. A non-transitory computer readable medium having storedthereon computer executable instructions for performing the method ofclaim
 1. 11. An apparatus for detecting a sample using the method ofclaim
 1. 12. A chromatographic apparatus for detecting and collectingone or more components within a first sample stream comprising: achromatography column in fluid communication with the first samplestream; a second sample stream comprising a carrier gas; a splitter thatmoves a portion of the first sample stream to a second sample stream,which is in fluid communication with at least one destructive detectorto generate at least one response value during a chromatographic run; aprocessor that is adapted to receive said at least one response valueand to generate at least one signal from the at least one response valuereceived from said destructive detector; and a fraction collectoradapted to collect at least one fraction corresponding to at least oneof said components in response to a change said at least one signal. 13.The apparatus of claim 12, wherein said fraction is collected during thechromatographic run.
 14. The apparatus of claim 12, including at leastone destructive detector selected from the group consisting ofevaporative light scattering detectors (ELSD), mass spectrometers (MS),condensation nucleation light scattering detectors (CNLSD), and coronadischarge detectors.
 15. The apparatus of claim 12, wherein saidchromatography system further comprises at least one non-destructivedetector selected from the group consisting of optical absorbancedetectors, refractive index detectors (RID), fluorescence detectors(FD), chiral detectors (CD), and conductivity detectors.
 16. Theapparatus of claim 15, wherein the non-destructive detector includes atleast one optical absorbance detector.
 17. The apparatus of claim 12,herein the destructive detector comprises an evaporative lightscattering detector.
 18. The apparatus of claim 12, wherein saidsplitter comprises a splitter pump; a shuttle valve; or a combination ofa splitter pump and a shuttle valve; wherein the splitter pump, theshuttle valve, or the combination are in fluid communication with the atleast one destructive detector.